Download Review Severe acute respiratory syndrome coronavirus entry as a

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Antiviral Therapy 12:639–650
Review
Severe acute respiratory syndrome coronavirus
entry as a target of antiviral therapies
Jens H Kuhn1,2, Wenhui Li1, Sheli R Radoshitzky1, Hyeryun Choe 3 and Michael Farzan1*
1
Department of Microbiology and Molecular Genetics, Harvard Medical School, New England Primate Research Center, Southborough,
MA, USA
2
Department of Biology, Chemistry, Pharmacy, Freie Universität Berlin, Berlin, Germany
3
Department of Pediatrics, Children’s Hospital, Harvard Medical School, Boston, MA, USA
*Corresponding author: Tel: +1 508 624 8019; Fax: +1 508 786 3317; E-mail: [email protected]
The identification in 2003 of a coronavirus as the aetiological agent of severe acute respiratory syndrome (SARS)
intensified efforts to understand the biology of coronaviruses in general and SARS coronavirus (SARS-CoV) in
particular. Rapid progress was made in describing the
SARS-CoV genome, evolution and lifecycle. Identification
of angiotensin-converting enzyme 2 (ACE2) as an
obligate cellular receptor for SARS-CoV contributed to
understanding of the SARS-CoV entry process, and
helped to characterize two targets of antiviral therapeutics:
the SARS-CoV spike protein and ACE2. Here we describe
the role of these proteins in SARS-CoV replication and
potential therapeutic strategies aimed at preventing
entry of SARS-CoV into target cells.
Introduction
The viral order Nidovirales currently contains the three
families Arteriviridae, Coronaviridae, and Roniviridae
[1,2]. The viruses assigned to these families differ in
morphology but have a similar genomic organization.
The most prominent shared feature of the nidoviruses is
transcription of a set of nested, subgenomic messenger
RNAs [1,2]. Human pathogens are found exclusively
among the two genera Coronavirus and Torovirus in
the family Coronaviridae [2,3].
Coronavirions (genus Coronavirus) contain a single
copy of a 28- to 31-kb-long, capped and polyadenylated,
linear, single-stranded RNA of positive polarity, which is
helically encapsidated by nucleocapsid (N) proteins and
surrounded by membrane (M) proteins. The envelope
contains protrusions (spike [S] proteins) that are major
antigenic determinants of coronavirions, which are
100–120 nm in diameter [4]. Virions attach to cellsurface receptors via their spike proteins, which then
mediate fusion with the host cell membrane. Genome
replication occurs in the cytoplasm. After replication and
maturation, coronaviruses bud from the endoplasmic
reticulum–Golgi intermediate compartment [4].
Coronaviruses infect a wide spectrum of animal
species, including many different mammals and birds in
which they cause acute or chronic upper respiratory,
© 2007 International Medical Press 1359-6535
gastrointestinal, hepatic, or central nervous system
diseases. Classically, three distinct genetic and serological
coronavirus groups are differentiated [4]. Human
coronavirus 229E (HCoV-229E, group 1) and HCoVOC43 (group 2) are the aetiological agents of mild and
usually self-resolving upper respiratory tract infections in
otherwise healthy people or severe pneumonias in
immunocompromised individuals [3]. Several pathogenic
human coronaviruses (B814, HCoV-OC16, HCoVOC37 and HCoV-OC48) await characterization [3].
Emerging coronaviruses
In recent years, three more pathogenic human
coronaviruses have been identified.
In 2003, a novel coronavirus, severe acute respiratory
syndrome coronavirus (SARS-CoV), was identified as the
aetiological agent of SARS [5–9], which had first
emerged in November of 2002 in Guangdong Province,
China. Infected people presented with an influenza-like
disease beginning with dyspnoea, headaches, myalgia
and pyrexia, followed by acute pneumonia and respiratory failure. SARS-CoV transmits via droplets, fomites
and direct person-to-person contact, which is one of the
reasons why SARS evolved into a pandemic. A total of
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JH Kuhn et al.
8,096 cases and 774 deaths (lethality 9.6%) were
recorded in Asia, Europe and North America before the
outbreak was declared over after successful public health
intervention in July of 2003 [10–14]. A second emergence
of SARS was recorded during the winter of 2003–2004,
again in Guangdong Province. During this limited
outbreak, only four individuals developed the disease and
all survived [15–17]. Since then, SARS-CoV seems to
have disappeared because no other human cases, with the
exception of laboratory infections [18,19], have been
identified. The natural reservoir of SARS-CoV remains
elusive. Animals kept and sold at a Guangdong market
place are likely to have been the immediate origin of
the virus found in humans; market Chinese ferret
badgers (Melogale moschata), Himalayan palm civets
(Paguma larvata) and raccoon dogs (Nyctereutes
procyonoides) were later shown to harbour viruses
similar to SARS-CoV [20]. Himalayan palm civets are
of special interest because (i) SARS-CoV can persist in
Himalayan palm civets for weeks [21], (ii) the infections
of 2003–2004 were connected to restaurants that
served Himalayan palm civet meat [16,17], and (iii)
culling of Himalayan palm civets and their removal
from markets dramatically reduced SARS case
numbers. However, it is unlikely that the Himalayan
palm civet or other marketplace animals serve as natural
reservoirs for the virus. Only some marketplace
Himalayan palm civets tested positive for SARS-CoV,
whereas animals from farms and captured in the wild
were found to be free of infection [22,23]. Also, molecular studies imply that the SARS-CoV genome was not at
equilibrium in the Himalayan palm civet host [17,22]. In
the past 2 years, numerous novel full and partial coronaviral genomes from all coronaviral groups have been
obtained from bats [24–31], including SARS-CoV-like
viruses from Chinese horseshoe bats (Rhinolophus spp.)
[27,29–31]. The isolated viruses are genetically diverse
and do not appear to cause disease in the bat hosts,
suggesting that the precursor of SARS-CoV is
harboured by a yet-unidentified bat species and was
transmitted to Himalayan palm civets. In the laboratory,
SARS-CoV isolates from the 2002–2003 epidemic
infect cats [32], ferrets [32], hamsters [33] and
cynomolgus and rhesus macaques [34,35], and can be
adapted to infect mice [36–39]. Although these animals
spontaneously clear infection [21,32] and transmission
has only been observed among cats and ferrets [32],
these observations suggest that SARS-CoV could readily
jump species barriers.
SARS-CoV found in humans and Himalayan palm
civets and SARS-CoV-like genomes from bats are
outliers of group 2 coronaviruses. For this reason the
classical division of the genus Coronavirus has been
questioned. Some researchers classify these novel
viruses as a subgroup (group 2b), whereas others
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consider them representatives of a novel group 4. Some
of the recently discovered novel bat coronaviruses
unrelated to SARS-CoV and SARS-CoV-like viruses
have been assigned to a putative group 5
[24,25,40–43]. However, none of these new groups
have yet been accepted by the International Committee
on Taxonomy of Viruses.
Two other human coronaviruses have been identified
since the discovery of SARS-CoV. HCoV-NL63, a group
1 virus, has been implicated in conjunctivitis, croup and
respiratory infections in children. Recent surveys suggest
that this virus is distributed globally [44–52]. A third
novel human coronavirus, HCoV-HKU1 (group 2), was
isolated from a 71-year-old man with pneumonia [53],
and has since been found in patients with respiratory
diseases in many countries, including Australia [54],
France [55] and the United States [56].
Coronavirus cell entry
The cell, tissue and host tropism of coronaviruses, as
well as their virulence, is mainly determined by the S
proteins, which protrude from the virion as large and
distinct club- or petal-shaped, 20–40-nm-long
peplomers [57–62], and, in some cases, also by the
haemagglutin–esterase (HE) proteins [4]. S proteins
are class I fusion and type 1 transmembrane proteins
[63,64]. They consist of a large ectodomain, a transmembrane anchor, and a short cytoplasmic tail. The
ectodomain contains distinct N-terminal (S1) and Cterminal (S2) domains. S1 binds to the host cellsurface receptor via a distinct, independently folded
receptor-binding domain (RBD) [65,66], in some
cases inducing conformational changes that expose a
fusion peptide embedded in S2. This in turn results in
a reorganization of S2’s two heptad repeats (HR-1
and HR-2) into coiled coils [64,67], followed by
fusion of the cellular plasma membrane and the viral
envelope, thereby releasing the coronaviral nucleocapsid into the cytoplasm [64]. Prior to virion
budding, S proteins are either cleaved between their
S1 and S2 domains by a furin-like protease in the Sprotein-expressing and virus-producing cell before
the S protein becomes incorporated into the budding
virion (for instance, infectious bronchitis virus and
murine hepatitis virus) [68,69], or remain uncleaved
before incorporation into virions (for example,
HCoV-229E, HCoV-NL63 and SARS-CoV) [70–72].
After receptor binding, SARS-CoV, but not HCoVNL63, is dependent on S protein cleavage by
cathepsin L or S in an endosomal or lysosomal
compartment or by exogenous proteases such as
elastase, thermolysin or trypsin, which target a
limited number of S protein cleavage sites [73–75].
Whereas S protein cleavage within virus-producing
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SARS–ACE2 interaction
cells is now understood to be a step in the activation
of the membrane-fusion activity of the S2 domain,
the function of lysosomal cathepsin cleavage remains
to be determined.
Several coronavirus cell-surface receptors are known
(Table 1). The zinc metalloprotease aminopeptidase N
(APN, CD13) is the receptor of the group 1 viruses
canine coronavirus, feline infectious peritonitis virus,
HCoV-229E, porcine transmissible gastroenteritis virus
and porcine epidemic diarrhea virus [66,76–79], but is
not a receptor for HCoV-NL63 [80]. The receptor of
HCoV-OC43 remains elusive, but the receptors of
other group 2 viruses have been identified. For
instance, those of murine hepatitis virus and bovine
coronavirus have been identified as members of the
carcinoembryonic antigen-cell adhesion family and 9O-acetylated sialic acids, respectively [81,82].
Receptors for group 3 (avian coronaviruses) and putative
group 5 viruses have not yet been identified.
Identification of human ACE2 as a SARSCoV and HCoV-NL63 receptor
Analyses of the SARS-CoV S protein revealed a relatively
low (20–27%) amino acid sequence identity to the
spikes of other coronaviruses [83,84]. Although SARSCoV spikes are not cleaved by a host cell protease into
S1 and S2 subunits prior to budding, discrete SARSCoV S1 and S2 domains can be delineated by similarity
to the subunits of cleaved S proteins [72,83,85]. Using
the S1 domain, angiotensin-converting enzyme 2
(ACE2) was identified as the principal SARS-CoV
receptor after immunoprecipitation from lysates of
SARS-CoV-susceptible African green monkey kidney
(Vero E6) cells and mass spectrometry analysis [86].
Following cellular overexpression, recombinant human
ACE2 supported the formation of syncytia with Sprotein-expressing human embryonic kidney (HEK
293T) cells. A soluble form of ACE2, and anti-ACE2
Table 1. Viruses of the genus Coronavirus (Nidovirales: Coronaviridae) and their receptors, modified from [4]
Classical
subgroup
Species
Virus (abbreviation)
Receptor
1
1
Canine coronavirus
Feline coronavirus
1
1
1
1
Human coronavirus 229E
‘Human coronavirus NL63’
Porcine epidemic diarrhoea virus
Transmissible gastroenteritis virus
2
Bovine coronavirus
Canine coronavirus (CCoV)
Feline coronavirus (FCoV)
Feline infectious peritonitis virus (FIPV)
Human coronavirus 229E (HCoV-229E)
‘Human coronavirus NL63 (HCoV-NL63)’
Porcine epidemic diarrhoea virus (PEDV)
Porcine respiratory coronavirus (PRCoV)
Transmissible gastroenteritis virus (TGEV)
Bovine coronavirus
2
Human coronavirus OC43
Human coronavirus OC43
2
2
2
2
‘Human coronavirus HKU1’
Human enteric coronavirus
Murine hepatitis virus
Porcine haemagglutinating
encephalomyelitis virus
Puffinosis coronavirus
Rat coronavirus
‘Human coronavirus HKU-1 (HCoV-HKU1)’
Human enteric coronavirus (HECoV)
Murine hepatitis virus (MHV)
Porcine haemagglutinating
encephalomyelitis virus (HEV)
Puffinosis coronavirus (PCoV)
Rat coronavirus (RtCoV))
Sialodacryoadenitis virus (SDAV)
Severe acute respiratory syndrome
coronavirus (SARS-CoV)
Infectious bronchitis virus (IBV)
Pheasant coronavirus (PhCoV)
Turkey coronavirus (TCoV)
‘B814’
‘Human coronavirus OC16’
‘Human coronavirus OC37’
‘Human coronavirus OC48’
Rabbit coronavirus (RbCoV)
APN
APN
APN
APN
ACE2
APN
APN
APN
N-acetyl-9-Oacetylneuraminic acid?
N-acetyl-9-Oacetylneuraminic acid?
?
?
CEACAMs
?
2
2
2
3
3
3
NC
NC
NC
NC
NC
Severe acute respiratory
syndrome coronavirus
Infectious bronchitis virus
Pheasant coronavirus
Turkey coronavirus
?
?
?
?
Rabbit coronavirus
?
?
?
ACE2
N-Acetylneuraminic acid?
?
?
?
?
?
?
?
Names not listed by the International Committee on Taxonomy of Viruses (ICTV) are marked by inverted commas. ACE2, angiotensin-converting enzyme 2; APN,
aminopeptidase N (CD13); CEACAMs, carcinoembryonic antigen-related cell adhesion molecules; L-SIGN, liver/lymph-node-specific intercellular adhesion molecule-3grabbing integrin (CD209L, DC-SIGN2, DC-SIGNR); NC, not classified/tentative.
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antisera, but not anti-ACE1 antisera, inhibited SARSCoV replication in Vero E6 cells. Finally, human ACE2
expression in otherwise SARS-CoV-resistant cells
rendered them permissive to transduction with
lentiviruses pseudotyped with S protein [71,86–88].
ACE2 was also identified independently after transduction of HeLa cells with a retrovirus cDNA library from
Vero E6 cells, and flow-cytometry-based selection of
cells that bound to a purified S protein fragment [89].
Additional surface proteins might contribute to efficient
SARS-CoV cell entry. For instance, DC-SIGN (CD209),
DC-SIGNR (L-SIGN, CD209L) and LSECtin enhanced
infection of ACE2-expressing cells [90–93]. However,
these molecules did not mediate infection in the absence
of ACE2, suggesting that they are attachment factors
rather than true alternative receptors. ACE2 is currently
the only known SARS-CoV receptor and is likely to be
central to replication of virus in infected animals. This
latter assertion is supported by multiple lines of
evidence. First, human ACE2 binds the SARS-CoV S1
domain specifically and with high affinity (1.7 nM) [94].
Second, all cell lines supporting efficient SARS-CoV
infection express ACE2 [87,95,96]. Third, the efficiency
of SARS-CoV infection in cats, chickens, dogs, ferrets,
hamsters, Himalayan palm civets, humans, mice, minks,
monkeys and rats, or their cells, directly correlates with
the ability of the various ACE2 orthologues to promote
SARS-CoV cell entry [97–102]. Fourth, SARS-CoV S
protein and S protein mRNA were only detected in
ACE2-expressing cells in the lungs and gastrointestinal
tract of humans, which are major sites of SARS-CoV
replication [103–107]. Fifth, the S protein ACE2-binding
domain, its RBD, induces neutralizing antibodies in
mice, and anti-S-protein antibodies that inhibit ACE2-Sprotein association protect hamsters and mice from
SARS-CoV infection [108–115]. Finally, SARS-CoV
cannot replicate in ACE2–/– mice, but transgenic mice
expressing human ACE2 die from a SARS-like disease
after infection [116,117].
ACE2 is a carboxy-metalloprotease that contains a
single active site with a HEXXH zinc-binding motif.
Notably, APN, the receptor of most group 1 coronaviruses, is also a zinc-binding metalloprotease
[118–120]. The crystal structure of ACE2 demonstrates
a claw-like structure. An open–closed conformational
change of the claw is triggered upon ligand binding
[121]. By cleaving a variety of regulatory peptides
mainly involved in blood pressure homeostasis,
including the potent vasoconstrictor angiotensin II,
ACE2 appears to counterbalance the actions of the
related molecule ACE, which cleaves the inactive
peptide angiotensin I to the active angiotensin II [122],
and which cannot function as a SARS-CoV receptor
[87]. Furthermore, severe heart contractility defects
were observed in mice after targeted disruption of
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ACE2 [123,124]. The enzymatic activity of ACE2 does
not contribute to its ability to function as a SARS-CoV
receptor. Small molecule catalytic inhibitors did not
disturb ACE2–S protein interaction, and even S protein
bound to ACE2 did not alter the catalytic activity of the
enzyme [97]. This observation is in accordance with
results obtained for other coronaviruses. For instance,
cell entry of the group 1 coronavirus porcine transmissible gastroenteritis virus is also independent of the
proteolytic activity of its receptor, APN [125]. On the
other hand, ACE2 proteolysis or S-protein-mediated
ACE2 downregulation has been suggested to have a role
in SARS pathogenesis [126].
ACE2 and the SARS-CoV and HCoV-NL63 RBDs
A 193-amino-acid S1 fragment (residues 318–510) was
identified as the SARS-CoV RBD (Figure 1). This
protein fragment, which is distinct in structure and
location from the RBDs of, for example, HCoV-299E
and murine hepatitis virus, binds human ACE2 with
higher affinity than full-length S1 [72,94,127]. The
crystal structure of the SARS-CoV RBD revealed that it
consists of two subdomains. The first subdomain, a
five-stranded antiparallel β-sheet with three connecting
α-helices, makes up the core of the RBD and is similar
to analogous regions of other group 2 coronaviruses.
The second subdomain is unique to SARS-CoV. This
extended loop (residues 424–494), termed the receptorbinding motif (RBM), represents a gently curved surface
(Figure 1) [128]. The base of this surface, a twostranded antiparallel β-sheet, cradles the N-terminal
helix of ACE2 directly; one ridge of the surface contacts
one ACE2 loop, whereas the other inserts between two
different ACE2 loops of the N-terminal lobe, which is
located away from the active site of the enzyme [128].
Curiously, HCoV-NL63 and SARS-CoV share the
same human tissue tropism. Albeit with lower affinity,
HCoV-NL63 also uses ACE2 as its main host cell
receptor [80,129] despite the fact that HCoV-NL63
and SARS-CoV S proteins share no amino acid identity
[130]. In fact, HCoV-NL63 S protein is related to that
of HCoV-229E (56% sequence identity), which uses
APN as a receptor. Mapping of the HCoV-NL63 RBD
indicates that this RBD spans a broader and less
discrete portion of the S1 domain [130].
ACE2 from other species
The possibility of several different animal species as
SARS-CoV hosts (among others, bats, Chinese ferret
badgers, Himalayan palm civets and raccoon dogs)
[20,24–31], the differences in susceptibility of
different animals and cells to SARS-CoV infection
[32–38,100], and the fact that certain SARS-CoV© 2007 International Medical Press
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SARS–ACE2 interaction
Figure 1. The SARS-CoV RBD complexed to human ACE2
Conserved in
group 2
SARS-CoV
RBD
RBM
ACE2
The receptor-binding domain (RBD) has two distinctive domains, one conserved among group 2 coronaviruses, and the receptor-binding motif (RBM). The RBM is in
direct contact with angiotensin-converting enzyme 2 (ACE2) and is apparently unique to severe acute respiratory syndrome coronavirus (SARS-CoV). Residues of the RBD
and a critical RBM residue, threonine 487, are shown as dark spheres. This residue, a serine in most SARS-CoV isolates from Guangdong marketplace animals, is a threonine in all SARS-CoV isolates from the 2002–2003 SARS outbreak. This threonine confers high-affinity binding to human and palm civet ACE2. It is in direct contact with
human ACE2 lysine residue 353 (shown as light spheres), which, in mutagenesis studies, has been found to be critical to the ACE2 role as a SARS-CoV receptor.
neutralizing antibodies inhibit only certain viral strains
[110] implied that there are significant ACE2 differences
among species and the RBDs of different viral isolates.
For instance, the ACE2 tissue distribution is comparable
in humans, mice and rats. Nevertheless, murine ACE2
bound less efficiently to the SARS-CoV and HCoVNL63 S1 domains and supported S-protein-mediated
pseudotype transduction less efficiently than human
ACE2 [98,130]. Murine NIH 3T3 cells supported
SARS-CoV replication one order of magnitude less efficiently than NIH 3T3 cells expressing human ACE2
[98]. Rat ACE2 did not support transduction with
SARS-CoV S protein pseudotypes at all. However, the
exchange of four amino acid residues of rat ACE2 for
the equivalent residues found in human ACE2 yielded
an efficient SARS-CoV receptor [98]: rat ACE2
residues 82–84 form a glycosylation site that is not
present on Himalayan palm civet, mouse, and human
Antiviral Therapy 12:4 Pt B
ACE2, and residue 353 is a histidine in mouse and rat
ACE2 but a lysine in Himalayan palm civet and human
ACE2. Additional comparisons of ACE2 orthologues
from other species such as cat, chicken, dog, hamster,
macaques and mink further emphasized that the SARSCoV susceptibility of each species is directly correlated
to individual crucial amino acid changes rather than
the overall relatedness of all orthologues to each other
[99,101]. Interestingly, ACE2 residue 353 is also lysine in
cats, chicken, cows, dogs, macaques, mink and pigs, but
some of these ACE2s (for example chicken) are inefficient
SARS-CoV receptors nevertheless [99,101]. Therefore,
post-translational modifications and additional
particular residues of ACE2 might also influence
SARS-CoV species sensitivity.
S proteins from SARS-CoV isolates obtained in
2002–2003 (TOR2 isolate) and in 2003–2004 (GD03
isolate), and from SARS-CoV-like viruses from
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Himalayan palm civets (SZ3 isolate) all bound
Himalayan palm civet ACE2. However, GD03 and SZ3
proteins bound human ACE2 less efficiently than TOR2
S protein [97]. Again, exchanging specific residues of
human ACE2 for those of Himalayan palm civet ACE2
enhanced binding of GD03 and SZ3 S proteins.
Interestingly, amino acid sequence comparisons of the
RBDs of SARS-CoV and SARS-CoV-like viruses
revealed only few strain variations [110]. Swapping of
RBD residues 479 and 487 of GD03 and SZ3 viruses
for the equivalent residues of TOR2 virus increased the
affinity for human ACE2 [97], but these exchanges had
no effect on the affinity to mouse ACE2 [131]. Residue
479 is an asparagine or serine in all SARS-CoV strains
isolated from humans, but a lysine in viruses isolated
from Himalayan palm civets or raccoon dogs. This lysine
is incompatible with human ACE2 binding, whereas
palm civet ACE2 can efficiently bind S proteins
expressing either asparagine or lysine [97]. S protein
residue 487 is a threonine in all SARS-CoV isolates of the
2002–2003 SARS outbreak [132], but a serine in the
isolates from the mild 2003–2004 outbreak and in almost
all SARS-CoV-like viruses from palm civets and raccoon
dogs. The change to serine resulted in an approximately
20-fold decrease in binding to human ACE2 [97]. An
increase in binding resulted when a threonine was
introduced into the SZ3 RBD. A threonine at position
487 also substantially increased association with
Himalayan palm civet ACE2 [97]. Taken together, these
data imply that only a few residues of the SARS-CoV
RBD and the host species ACE2 determine the efficiency
of S1–ACE2 interactions, that the observed lack of
severity and transmission of the 2003–2004 SARS
outbreak could be due to incomplete adaptation of
GD03 viruses to human ACE2, and that Himalayan
palm civets might be an important intermediate in the
transfer of SARS-CoV to humans.
SARS-CoV-like viruses from bats lack the RBM in
the RBD-equivalent region, including most residues
associated with ACE2 binding. This fact might
explain the inability of these viruses to replicate in
SARS-CoV-permissive cells [29,30,128], and suggests
that bat SARS-CoV-like viruses do not use ACE2 as a
host-cell receptor. These viruses might have acquired
the RBM, perhaps through recombination with a
HCoV-NL63-like virus in a Himalayan palm civet,
before evolving into SARS-CoV.
Host cell proteases involved in coronavirus
cell entry
The S proteins of several coronaviruses (for example,
HCoV-OC43) are cleaved between their S1 and S2
domains by furin-like proteases in the producer cell
prior to virion budding. In accordance with the
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biosynthesis of class I fusion proteins of viruses
belonging to other families (including retroviruses and
orthomyxo- viruses), this cleavage is understood to be
an activation step for the S2 domain to expose its
fusion peptide, usually at the N-terminus. However,
there are exceptions. For instance, filoviral spike
proteins, which are also class I fusion proteins, remain
fully functional even if the furin cleavage site is
knocked out [133]; the S protein of the group 2 coronavirus murine hepatitis virus remains cleaved or
uncleaved depending on cell type [134]; and various
other coronaviruses (including all group 1 coronaviruses and SARS-CoV) do not possess a furin-like
cleavage site at all. Additionally, the efficient cell entry
of some coronaviruses (murine hepatitis virus type 2
and SARS-CoV), but not of others (HCoV-NL63), is
dependent on proteolytic cleavage by lysosomal cysteine
proteases (cathepsins L and S, but not B in the case of
SARS-CoV) after the receptor-binding step [74,75,135]
and is sensitive to lysosomotropic agents such as
ammonium chloride or the proton ATPase inhibitor
bafilomycin A. Exogenous trypsin or thermolysin can
enhance SARS-CoV cell entry when added to virions
already attached to their cell-surface receptors. This
treatment also bypassed the entry inhibition induced by
ammonium chloride and general or specific cathepsin
inhibitors [73–75]. The true function of cathepsins in
the SARS-CoV and murine hepatitis virus type 2 life
cycles therefore remains to be determined.
Therapeutic entry inhibition
Potential targets for specific antivirals against SARS-CoV
and HCoV-NL63 include the coronaviral spike proteins
and their cognate receptor ACE2, and the coronaviral
proteases,
mRNA
cap-1
methyl-transferases,
NTPases/helicases
and
transcriptase–replicase
complexes [136–138]. HIV-1 protease inhibitors,
interferons and ribavirin were used empirically to treat
SARS during the two recognized outbreaks, but the
beneficial effect of these treatments, if any, is unclear
[139], although interferons at least were effective in
inhibiting SARS-CoV replication in tissue culture [136],
and prophylactic treatment of SARS-CoV-infected
cynomolgus macaques with pegylated interferon-α
significantly reduced viral replication and excretion
[140]. At the time of writing, there are still no specific
treatments of proven value available to prevent or
treat SARS or HCoV-NL63 infections. However,
several promising vaccine candidates are now being
evaluated. The rapid molecular characterization of
the cell-entry determinants of both agents and the
development of a mouse and non-human primate
model for SARS suggest that the development and
evaluation of an effective treatment regimen should be
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SARS–ACE2 interaction
possible in the near future. Any such treatment would
have the advantage of neutralizing the viruses before
they attach to a host cell or during the entry step itself,
thereby possibly preventing the need for drug delivery
into target cells.
Several entry-specific approaches have been
pursued. Sera of convalescent SARS patients contain
high titres of SARS-CoV-neutralizing antibodies, and
the administration of the sera themselves proved to be
beneficial to acutely ill SARS patients [141,142].
These observations suggested that the virus itself
could be neutralized by specific antibodies that
prevent its binding to its receptor. Indeed, several
studies have identified the SARS-CoV RBD to be a
major immune response determinant that contains
multiple
important
neutralizing
epitopes
[108,114,115,143–148], supporting the idea that
virus neutralization occurs mainly by interruption of
the S protein–ACE2 interaction. In contrast to many
other viruses, the SARS-CoV RBD is exposed on,
rather than hidden from, the surface of the S protein,
perhaps reflecting the fact that SARS-CoV favours
rapid
transmission
over
immune
escape.
Consequently, neutralizing antibodies to the RBD
are easily induced even if full-length S protein is used
as an inoculum [109,147]. For instance, a recombinant human single-chain variable region fragment
against the SARS-CoV (TOR2 isolate) S1 domain
from non-immune human antibody libraries (80R
scFV) inhibited the formation of syncytia between
ACE2-expressing and S-protein-expressing HEK 293
T-cells, and efficiently neutralized the infection of
Vero E6 cells with SARS-CoV (Urbani isolate of the
2002–2003 epidemic). 80R scFV competed with
soluble ACE2 for association with the S1 domain, and
bound S1 with high affinity (Kd=32.3 nM). A human
immunoglobulin G1 (IgG1) form of 80R scFV (80R)
bound S1 with higher affinity (Kd=1.59 nM) and
neutralized SARS-CoV (Urbani isolate) with a 20-fold
higher efficiency than 80R scFV (IC50=0.37 nM)
[109]. 80R binds to a conformationally sensitive S
protein fragment (residues 324–503) that is located
within the RBD and precipitates a fusion protein
consisting of the RBD and the Fc region of human IgG1
(RBD–Fc) as efficiently as protein A [110]. Just like
other monoclonal antibodies that react with the RBD,
80R inhibited SARS-CoV replication in mice at doses
usable in humans [108,110,144]. However, it is
important to note that 80R does not neutralize all
SARS-CoV strains. For instance, the GD03 isolate is
totally resistant to 80R because of a D480G mutation
that destroys the 80R epitope [110]. Another study
employed the RBD as a subunit vaccine. RBD–Fc in
combination with Freud’s complete adjuvant induced
antibodies in intradermally immunized New Zealand
Antiviral Therapy 12:4 Pt B
white rabbits that completely inhibited infection of
Vero E6 cells with 100× 50% tissue culture infective
doses of SARS-CoV at a serum dilution of 1:10,240.
These antibodies also inhibited the interaction of S1 with
commercially available ACE2 [111]. The immunization
of mice and rabbits with inactivated SARS-CoV
induced antibodies that specifically recognized the
RBD, inhibited the ACE2–RBD interaction and
prevented cell transduction with S-protein-pseudotyped
lentiviruses [149]. SARS-CoV-neutralizing antibodies
recognizing the RBD could also be induced in
BALB/c mice, rabbits and Chinese rhesus macaques
by immunization with attenuated, modified vaccinia
virus Ankara expressing the SARS-CoV Urbani isolate
S protein [143]. However, one important safety
concern in treating SARS patients with reconvalescent
sera, monoclonal antibodies or antibody-inducing
viral fragments is antibody-dependent enhancement
(ADE) of infection (for a review see [150]). For
instance, ADE has been observed in feline peritonitis
virus entry into primary feline macrophages [151], and
antibodies that neutralized entry of lentiviruses
pseudotyped with S protein from human SARS-CoV
isolates enhanced cell entry of lentiviruses pseudotyped
with S protein derived from plam civet SARS-CoV
isolates [152].
In addition to the use of antibodies, the RBD itself, or
modified peptides thereof, could be used as a therapeutic
because the RBD competitively inhibits virion binding to
ACE2. In vitro, RBD–Fc inhibited transduction of
ACE2-overexpressing target cells with lentiviruses
pseudotyped with SARS-CoV Urbani isolate S protein
with a lower 50% inhibitory concentration (IC50;
10 nM) than S1–Fc (50 nM) [94]. In a mouse model,
RBD–Fc elicited high titres of long-lasting neutralizing
antibodies and protected the animals from challenge
with infectious virus [153]. Also, an RBD peptide,
consisting of residues 471–503, specifically inhibited
SARS-CoV cell entry in vitro [154].
Soluble, recombinant, enzymatically inactive ACE2,
or modified polypeptides containing its SARS-CoVbinding domain, could also be used as a decoy to
neutralize SARS-CoV in the bloodstream [95], because
it would bind to the virions’ peplomers, thereby
preventing binding to cell-surface ACE2. In vitro,
enzymatically inactive, soluble ACE2–Fc inhibited
lentiviral pseudotype transduction more efficiently than
S1–Fc, at an IC50 of 2 nM [71,97]. Furthermore, an
ACE2-derived peptide, consisting of residues 22–44 and
351–357 linked by a glycine, exhibited anti-SARS-CoV
activity with an IC50 of 100 nM [155].
Antibodies to ACE2 inhibited SARS-CoV replication
in Vero E6 cells with an IC50 of 1.5 µg/ml [86]. On
the basis of the available crystal structure of ACE2
complexed with the SARS-CoV RBD [128] it should
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also be possible to develop specific proteinaceous
inhibitors of ACE2 that do not contain RBD fragments
or even small molecules that could interrupt
ACE2–RBD interaction. At least one such inhibitor, a
molecule that prevents both binding of SARS-CoV to
ACE2 and ACE2 enzymatic activity, has been identified
[156]. Other small molecules, VE607 and Emodin,
inhibited SARS-CoV cell entry or pseudotype transduction in a dose-dependent manner, but it is not yet
clear whether the molecules act on S protein or on
ACE2 [157,158]. Because SARS-CoV binds to ACE2
at a location distinct from its active site [128] it
should be possible to find inhibitors that only
prevent SARS-CoV binding, but do not disturb the,
probably important, physiological function of ACE2.
Interestingly, both SARS-CoV and HCoV-NL63
use ACE2 as their receptors and both bind to overlapping but not identical sites away from ACE2’s
catalytic centre [80,86,89,130]. This fact could
permit the development of an inhibitor that targets
both viruses at the same time.
After binding to ACE2, SARS-CoV and HCoV-NL63
S proteins initiate fusion with the host cell membrane
following a mechanism similar to that induced by
other class I fusion proteins such as those of
filoviruses, orthomyxoviruses and retroviruses.
Experiments suggest that receptor binding occurs at
neutral pH and that no viral proteins other than the
spike proteins are required for this step and subsequent
fusion [72,86]. The conformational changes of S2’s
two heptad repeat regions HR-1 and HR-2 are critical
for efficient fusion. Therefore, inhibiting the HR1–HR-2 interaction might prove to be another
antiviral strategy. Indeed, synthetic peptides derived
from the SARS-CoV HR-2 region that bound efficiently
to HR-1 peptides interrupted the conformational
changes and inhibited SARS-CoV infection, albeit
with relatively low efficacy [159–162].
It could also be possible to develop SARS-CoVspecific antivirals based on host-cell protease inhibitors
since efficient SARS-CoV entry is dependent on the
activity of the lysosomal cysteine protease cathepsin L
or S [74,75]. For instance, cell transduction with
lentiviral particles pseudotyped with SARS-CoV S
protein was inhibited in vitro by leupeptin (a serine and
cysteine protease inhibitor), E64c (a cysteine protease
inhibitor) and Z-lll-FMK (a cathepsin B and L
inhibitor) at 95% inhibitory concentrations of 15.2,
8.2 and 3.5 µM, respectively [74,163].
Conclusions
It remains unclear whether a SARS-CoV-like virus
will re-emerge to threaten public health, as SARSCoV did in the winter of 2002–2003. However, if
646
such a virus is to emerge, it will likely have several
properties that might help to address the threat. First,
a dangerous coronavirus is likely to favour rapid
transmission over subtle means of immune escape,
and therefore subunit vaccination might be a safe and
effective way to safeguard against its widespread
transmission. The severity of SARS-CoV might also
be due in part to its high-affinity association with its
receptor, consistent with studies of both SARS-CoV
and other coronaviruses. If so, soluble receptor
mimetics might be effective in addressing an
outbreak, along with antibodies that bind the
receptor or the S protein. SARS-CoV depends on the
endosomal protease cathepsin L. Cathepsin L
inhibitors have been developed as anti-cancer therapies,
and might also be useful as antivirals. In short, there
are a number of tools that might be useful in
addressing the next severe coronavirus outbreak.
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Accepted for publication 9 February 2007
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